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L-selectin shedding does not regulate constitutive T cell trafficking but controls the migration pathways of antigen-activated T lymphocytes.

Galkina E, Tanousis K, Preece G, Tolaini M, Kioussis D, Florey O, Haskard DO, Tedder TF, Ager A - J. Exp. Med. (2003)

Bottom Line: WT, but not LDeltaP-selectin, underwent rapid, metalloproteinase-dependent shedding after TCR engagement, and LDeltaP T cells retained the capacity to enter PLNs from the bloodstream.These results suggest that the ability to shed L-selectin is not required for T cell recirculation and homing to PLNs.However, L-selectin shedding from antigen-activated T cells prevents reentry into PLNs.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Immunology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK.

ABSTRACT
L-selectin mediates rolling of lymphocytes in high endothelial venules (HEVs) of peripheral lymph nodes (PLNs). Cross-linking of L-selectin causes proteolytic shedding of its ectodomain, the physiological significance of which is unknown. To determine whether L-selectin shedding regulates lymphocyte migration, a mutant form that resists shedding (LdDeltaP-selectin) was engineered. Transgenic mice expressing either LDeltaP or wild-type (WT) L-selectin on T cells were crossed with L-selectin knockout (KO) mice. The cellularity and subset composition of secondary lymphoid organs did not differ between LDeltaP and WT mice, however, they were different from C57BL/6. Plasma levels of soluble L-selectin in LDeltaP mice were reduced to <5% of WT and C57BL/6 mice. The rolling properties of T lymphocytes from LDeltaP and WT mice on immobilized L-selectin ligands were similar. Furthermore, similar numbers of LDeltaP and WT T lymphocytes were recruited from the bloodstream into PLNs in mice, although LDeltaP T cells transmigrated HEVs more slowly. WT, but not LDeltaP-selectin, underwent rapid, metalloproteinase-dependent shedding after TCR engagement, and LDeltaP T cells retained the capacity to enter PLNs from the bloodstream. These results suggest that the ability to shed L-selectin is not required for T cell recirculation and homing to PLNs. However, L-selectin shedding from antigen-activated T cells prevents reentry into PLNs.

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MMP-dependent shedding of L-selectin after TCR engagement regulates entry into PLNs. (A) PLN lymphocytes from F5/RAG-1 KO, F5/WT, and F5/LΔP mice were incubated with 0.1 μm cognate NP-68 or control peptide or 300 nM PMA at 37°C for 60 min in the absence and presence of 30 μM Ro31-9790. L-Selectin expression on F5-positive CD8 T cells was determined by flow cytometric analysis of dual-stained cells. Results are pooled from at least three independent experiments and are the mean percentage of F5 T cells expressing L-selectin ± SE. (B) CFSE-labeled PLN lymphocytes from F5/WT mice were incubated with 0.1 μM NP-68 peptide at 37°C for 60 min, mixed with equal numbers of either naive WT or NP68-activated LΔP lymphocytes labeled with CMTMR, and injected into RAG-1 KO mice. The percentages of immigrant CFSE- and CMTMR-labeled cells in PLNs after 60 min are shown on the dot plots. Representative data from a single experiment are shown. (C) PLN lymphocytes from F5/WT and F5/LΔP mice were incubated with or without NP-68 peptide and peptide removed by washing. In some experiments, Ro31-9790 was included during antigen activation to inhibit L-selectin shedding and removed before trafficking experiments. The lymphocyte populations to be compared were labeled with either CFSE or CMTMR and equal numbers injected into RAG-1 KO mice. After 60 min, the numbers of labeled cells in PLNs, spleen, and blood were measured by flow cytometry. The normalized ratio of CFSE/CMTMR-labeled cells in each organ was calculated as described in Materials and Methods. Results show mean localization ratios ± SE as follows: activated WT, naive F5/WT (black bars, n = 7); activated F5/WT, activated F5/LΔP (light gray bars, n = 7); activated F5/WT, activated F5/WT in the presence Ro31-9790 (white bars, n = 4); and activated LΔP, naive F5/LΔP (dark gray bars, n = 5). *, P < 0.05. **, P < 0.001.
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fig8: MMP-dependent shedding of L-selectin after TCR engagement regulates entry into PLNs. (A) PLN lymphocytes from F5/RAG-1 KO, F5/WT, and F5/LΔP mice were incubated with 0.1 μm cognate NP-68 or control peptide or 300 nM PMA at 37°C for 60 min in the absence and presence of 30 μM Ro31-9790. L-Selectin expression on F5-positive CD8 T cells was determined by flow cytometric analysis of dual-stained cells. Results are pooled from at least three independent experiments and are the mean percentage of F5 T cells expressing L-selectin ± SE. (B) CFSE-labeled PLN lymphocytes from F5/WT mice were incubated with 0.1 μM NP-68 peptide at 37°C for 60 min, mixed with equal numbers of either naive WT or NP68-activated LΔP lymphocytes labeled with CMTMR, and injected into RAG-1 KO mice. The percentages of immigrant CFSE- and CMTMR-labeled cells in PLNs after 60 min are shown on the dot plots. Representative data from a single experiment are shown. (C) PLN lymphocytes from F5/WT and F5/LΔP mice were incubated with or without NP-68 peptide and peptide removed by washing. In some experiments, Ro31-9790 was included during antigen activation to inhibit L-selectin shedding and removed before trafficking experiments. The lymphocyte populations to be compared were labeled with either CFSE or CMTMR and equal numbers injected into RAG-1 KO mice. After 60 min, the numbers of labeled cells in PLNs, spleen, and blood were measured by flow cytometry. The normalized ratio of CFSE/CMTMR-labeled cells in each organ was calculated as described in Materials and Methods. Results show mean localization ratios ± SE as follows: activated WT, naive F5/WT (black bars, n = 7); activated F5/WT, activated F5/LΔP (light gray bars, n = 7); activated F5/WT, activated F5/WT in the presence Ro31-9790 (white bars, n = 4); and activated LΔP, naive F5/LΔP (dark gray bars, n = 5). *, P < 0.05. **, P < 0.001.

Mentions: F5/RAG KO mice in which all lymphocytes express a class I restricted TCR receptor for influenza virus A/NT/60/68 nucleoprotein on CD8+ T cells (29) were used in the first instance to study antigen receptor induced shedding of endogenous L-selectin. The cognate peptide, NP-68, induced rapid, MMP-dependent shedding of endogenous L-selectin from CD8+ T cells in F5/RAG KO within 60 min. PMA induced shedding to a similar extent as NP-68 whereas a peptide from HIV GAG protein that does not bind the F5 TCR did not induce shedding (Fig. 8) . WT and LΔP mice were crossed with F5/L-selectin KO mice and offspring selected for expression of F5 TCR and transgenic L-selectin. In F5/WT and F5/LΔP mice, PLNs comprised ∼85% CD8+/F5+/L-selectin+ T cells, ∼10% CD4+/L-selectin+ T cells, and <5% L-selectin− B cells (unpublished data). NP-68 induced MMP-dependent shedding of transgenic L-selectin from CD8+ T cells in F5/WT mice but not from 10% CD8−/L-selectin+ cells found in these mice (unpublished data). PMA induced shedding from all L-selectin expressing cells and, therefore, was slightly greater than that induced by NP-68 (Fig. 8 A). In F5/LΔP mice, the number of CD8+ T cells expressing L-selectin and the mean fluorescence intensity were not altered after incubation with NP-68 peptide (Fig. 8 and not depicted).


L-selectin shedding does not regulate constitutive T cell trafficking but controls the migration pathways of antigen-activated T lymphocytes.

Galkina E, Tanousis K, Preece G, Tolaini M, Kioussis D, Florey O, Haskard DO, Tedder TF, Ager A - J. Exp. Med. (2003)

MMP-dependent shedding of L-selectin after TCR engagement regulates entry into PLNs. (A) PLN lymphocytes from F5/RAG-1 KO, F5/WT, and F5/LΔP mice were incubated with 0.1 μm cognate NP-68 or control peptide or 300 nM PMA at 37°C for 60 min in the absence and presence of 30 μM Ro31-9790. L-Selectin expression on F5-positive CD8 T cells was determined by flow cytometric analysis of dual-stained cells. Results are pooled from at least three independent experiments and are the mean percentage of F5 T cells expressing L-selectin ± SE. (B) CFSE-labeled PLN lymphocytes from F5/WT mice were incubated with 0.1 μM NP-68 peptide at 37°C for 60 min, mixed with equal numbers of either naive WT or NP68-activated LΔP lymphocytes labeled with CMTMR, and injected into RAG-1 KO mice. The percentages of immigrant CFSE- and CMTMR-labeled cells in PLNs after 60 min are shown on the dot plots. Representative data from a single experiment are shown. (C) PLN lymphocytes from F5/WT and F5/LΔP mice were incubated with or without NP-68 peptide and peptide removed by washing. In some experiments, Ro31-9790 was included during antigen activation to inhibit L-selectin shedding and removed before trafficking experiments. The lymphocyte populations to be compared were labeled with either CFSE or CMTMR and equal numbers injected into RAG-1 KO mice. After 60 min, the numbers of labeled cells in PLNs, spleen, and blood were measured by flow cytometry. The normalized ratio of CFSE/CMTMR-labeled cells in each organ was calculated as described in Materials and Methods. Results show mean localization ratios ± SE as follows: activated WT, naive F5/WT (black bars, n = 7); activated F5/WT, activated F5/LΔP (light gray bars, n = 7); activated F5/WT, activated F5/WT in the presence Ro31-9790 (white bars, n = 4); and activated LΔP, naive F5/LΔP (dark gray bars, n = 5). *, P < 0.05. **, P < 0.001.
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fig8: MMP-dependent shedding of L-selectin after TCR engagement regulates entry into PLNs. (A) PLN lymphocytes from F5/RAG-1 KO, F5/WT, and F5/LΔP mice were incubated with 0.1 μm cognate NP-68 or control peptide or 300 nM PMA at 37°C for 60 min in the absence and presence of 30 μM Ro31-9790. L-Selectin expression on F5-positive CD8 T cells was determined by flow cytometric analysis of dual-stained cells. Results are pooled from at least three independent experiments and are the mean percentage of F5 T cells expressing L-selectin ± SE. (B) CFSE-labeled PLN lymphocytes from F5/WT mice were incubated with 0.1 μM NP-68 peptide at 37°C for 60 min, mixed with equal numbers of either naive WT or NP68-activated LΔP lymphocytes labeled with CMTMR, and injected into RAG-1 KO mice. The percentages of immigrant CFSE- and CMTMR-labeled cells in PLNs after 60 min are shown on the dot plots. Representative data from a single experiment are shown. (C) PLN lymphocytes from F5/WT and F5/LΔP mice were incubated with or without NP-68 peptide and peptide removed by washing. In some experiments, Ro31-9790 was included during antigen activation to inhibit L-selectin shedding and removed before trafficking experiments. The lymphocyte populations to be compared were labeled with either CFSE or CMTMR and equal numbers injected into RAG-1 KO mice. After 60 min, the numbers of labeled cells in PLNs, spleen, and blood were measured by flow cytometry. The normalized ratio of CFSE/CMTMR-labeled cells in each organ was calculated as described in Materials and Methods. Results show mean localization ratios ± SE as follows: activated WT, naive F5/WT (black bars, n = 7); activated F5/WT, activated F5/LΔP (light gray bars, n = 7); activated F5/WT, activated F5/WT in the presence Ro31-9790 (white bars, n = 4); and activated LΔP, naive F5/LΔP (dark gray bars, n = 5). *, P < 0.05. **, P < 0.001.
Mentions: F5/RAG KO mice in which all lymphocytes express a class I restricted TCR receptor for influenza virus A/NT/60/68 nucleoprotein on CD8+ T cells (29) were used in the first instance to study antigen receptor induced shedding of endogenous L-selectin. The cognate peptide, NP-68, induced rapid, MMP-dependent shedding of endogenous L-selectin from CD8+ T cells in F5/RAG KO within 60 min. PMA induced shedding to a similar extent as NP-68 whereas a peptide from HIV GAG protein that does not bind the F5 TCR did not induce shedding (Fig. 8) . WT and LΔP mice were crossed with F5/L-selectin KO mice and offspring selected for expression of F5 TCR and transgenic L-selectin. In F5/WT and F5/LΔP mice, PLNs comprised ∼85% CD8+/F5+/L-selectin+ T cells, ∼10% CD4+/L-selectin+ T cells, and <5% L-selectin− B cells (unpublished data). NP-68 induced MMP-dependent shedding of transgenic L-selectin from CD8+ T cells in F5/WT mice but not from 10% CD8−/L-selectin+ cells found in these mice (unpublished data). PMA induced shedding from all L-selectin expressing cells and, therefore, was slightly greater than that induced by NP-68 (Fig. 8 A). In F5/LΔP mice, the number of CD8+ T cells expressing L-selectin and the mean fluorescence intensity were not altered after incubation with NP-68 peptide (Fig. 8 and not depicted).

Bottom Line: WT, but not LDeltaP-selectin, underwent rapid, metalloproteinase-dependent shedding after TCR engagement, and LDeltaP T cells retained the capacity to enter PLNs from the bloodstream.These results suggest that the ability to shed L-selectin is not required for T cell recirculation and homing to PLNs.However, L-selectin shedding from antigen-activated T cells prevents reentry into PLNs.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Immunology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK.

ABSTRACT
L-selectin mediates rolling of lymphocytes in high endothelial venules (HEVs) of peripheral lymph nodes (PLNs). Cross-linking of L-selectin causes proteolytic shedding of its ectodomain, the physiological significance of which is unknown. To determine whether L-selectin shedding regulates lymphocyte migration, a mutant form that resists shedding (LdDeltaP-selectin) was engineered. Transgenic mice expressing either LDeltaP or wild-type (WT) L-selectin on T cells were crossed with L-selectin knockout (KO) mice. The cellularity and subset composition of secondary lymphoid organs did not differ between LDeltaP and WT mice, however, they were different from C57BL/6. Plasma levels of soluble L-selectin in LDeltaP mice were reduced to <5% of WT and C57BL/6 mice. The rolling properties of T lymphocytes from LDeltaP and WT mice on immobilized L-selectin ligands were similar. Furthermore, similar numbers of LDeltaP and WT T lymphocytes were recruited from the bloodstream into PLNs in mice, although LDeltaP T cells transmigrated HEVs more slowly. WT, but not LDeltaP-selectin, underwent rapid, metalloproteinase-dependent shedding after TCR engagement, and LDeltaP T cells retained the capacity to enter PLNs from the bloodstream. These results suggest that the ability to shed L-selectin is not required for T cell recirculation and homing to PLNs. However, L-selectin shedding from antigen-activated T cells prevents reentry into PLNs.

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Related in: MedlinePlus